Contributions
Abstract: EP380
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
The Nucleoporin 98 (NUP98) gene is recurrently involved in the formation of oncogenic fusion proteins in acute myeloid leukemia (AML). While more than 30 partner genes have been identified to participate in NUP98 gene fusions, NUP98-JARID1A is the most frequent NUP98-rearrangement in infant leukemia and is associated with particularly poor prognosis. As no targeted therapy exists, there is an urgent need for a better understanding of the molecular mechanisms of NUP98-fusion-protein-dependent leukemogenesis to enable the development of tailored treatments. We and others have shown that oncogenic NUP98-fusion proteins act as transcriptional regulators, but it is incompletely understood how NUP98-fusion proteins directly regulate gene expression to drive leukemia.
Aims
To unravel critical effectors of NUP98-fusion-driven leukemogenesis we aimed to identify direct transcriptional targets of NUP98-JARID1A and characterize their requirements for the development and maintenance of NUP98-JARID1A-expressing AML.
Methods
To investigate functional genetic dependencies we conducted a genome-scale CRISPR/Cas9 loss-of-function screen in a cellular model of NUP98-JARID1A-dependent AML. Next we reasoned that a detailed understanding of the underlying transcriptional circuitries is critical to understand NUP98-fusion-dependent gene regulation. Using a newly developed model for ligand-induced degradation of the NUP98-JARID1A protein via the degradation tag (dTAG) system and performing nascent RNA-seq (SLAM-seq), we measured immediate transcriptional changes upon acute NUP98-JARID1A degradation.
Results
Analysis of the CRISPR/Cas9 screen identified 2550 genes that are required for the proliferation and survival of a NUP98-JARID1A AML cells. With the newly established model for ligand-induced NUP98-JARID1A loss we achieved complete degradation of the fusion protein within one hour after ligand addition, leading to terminal differentiation and apoptosis of leukemia cells. Global analysis of nascent mRNA expression by SLAM-seq revealed that 45 genes were significantly downregulated upon NUP98-JARID1A degradation. By intersecting these candidates with the 2550 genes that were required for NUP98-JARID1A-AML cell growth, we identified 12 high-confidence essential direct targets of NUP98-JARID1A.
Conclusion
This approach allows the identification of functional genetic dependencies and direct transcriptional target genes of NUP98-JARID1A in AML. Multi-layered validations of high-confidence candidates in a variety of models, including primary patient samples, will provide new insights into the molecular mechanisms and transcriptional circuitries of NUP98-JARID1A-driven leukemia and might identify novel actionable gene targets.
Keyword(s): Acute myeloid leukemia, Fusion, NUP98, Transcriptional regulation
Abstract: EP380
Type: E-Poster Presentation
Session title: Acute myeloid leukemia - Biology & Translational Research
Background
The Nucleoporin 98 (NUP98) gene is recurrently involved in the formation of oncogenic fusion proteins in acute myeloid leukemia (AML). While more than 30 partner genes have been identified to participate in NUP98 gene fusions, NUP98-JARID1A is the most frequent NUP98-rearrangement in infant leukemia and is associated with particularly poor prognosis. As no targeted therapy exists, there is an urgent need for a better understanding of the molecular mechanisms of NUP98-fusion-protein-dependent leukemogenesis to enable the development of tailored treatments. We and others have shown that oncogenic NUP98-fusion proteins act as transcriptional regulators, but it is incompletely understood how NUP98-fusion proteins directly regulate gene expression to drive leukemia.
Aims
To unravel critical effectors of NUP98-fusion-driven leukemogenesis we aimed to identify direct transcriptional targets of NUP98-JARID1A and characterize their requirements for the development and maintenance of NUP98-JARID1A-expressing AML.
Methods
To investigate functional genetic dependencies we conducted a genome-scale CRISPR/Cas9 loss-of-function screen in a cellular model of NUP98-JARID1A-dependent AML. Next we reasoned that a detailed understanding of the underlying transcriptional circuitries is critical to understand NUP98-fusion-dependent gene regulation. Using a newly developed model for ligand-induced degradation of the NUP98-JARID1A protein via the degradation tag (dTAG) system and performing nascent RNA-seq (SLAM-seq), we measured immediate transcriptional changes upon acute NUP98-JARID1A degradation.
Results
Analysis of the CRISPR/Cas9 screen identified 2550 genes that are required for the proliferation and survival of a NUP98-JARID1A AML cells. With the newly established model for ligand-induced NUP98-JARID1A loss we achieved complete degradation of the fusion protein within one hour after ligand addition, leading to terminal differentiation and apoptosis of leukemia cells. Global analysis of nascent mRNA expression by SLAM-seq revealed that 45 genes were significantly downregulated upon NUP98-JARID1A degradation. By intersecting these candidates with the 2550 genes that were required for NUP98-JARID1A-AML cell growth, we identified 12 high-confidence essential direct targets of NUP98-JARID1A.
Conclusion
This approach allows the identification of functional genetic dependencies and direct transcriptional target genes of NUP98-JARID1A in AML. Multi-layered validations of high-confidence candidates in a variety of models, including primary patient samples, will provide new insights into the molecular mechanisms and transcriptional circuitries of NUP98-JARID1A-driven leukemia and might identify novel actionable gene targets.
Keyword(s): Acute myeloid leukemia, Fusion, NUP98, Transcriptional regulation